Forgotten memory storage and retrieval in Drosophila.

Inaccessibility of stored memory in ensemble cells through the forgetting process causes animals to be unable to respond to natural recalling cues. While accumulating evidence has demonstrated that reactivating memory-stored cells can switch cells from an inaccessible state to an accessible form and lead to recall of previously learned information, the underlying cellular and molecular mechanisms remain elusive. The current study used Drosophila as a model to demonstrate that the memory of one-trial aversive olfactory conditioning, although inaccessible within a few hours after learning, is stored in KCαß and retrievable after mild retraining. One-trial aversive conditioning triggers protein synthesis to form a long-lasting cellular memory trace, approximately 20 days, via creb in KCαß, and a transient cellular memory trace, approximately one day, via orb in MBON-α3. PPL1-α3 negatively regulates forgotten one-trial conditioning memory retrieval. The current study demonstrated that KCαß, PPL1-α3, and MBON-α3 collaboratively regulate the formation of forgotten one-cycle aversive conditioning memory formation and retrieval.

Aversive conditioning information transmission in Drosophila.

Animals rapidly acquire surrounding information to perform the appropriate behavior. Although social learning is more efficient and accessible than self-learning for animals, the detailed regulatory mechanism of social learning remains unknown, mainly because of the complicated information transfer between animals, especially for aversive conditioning information transmission. The current study revealed that, during social learning, the neural circuit in observer flies used to process acquired aversive conditioning information from demonstrator flies differs from the circuit used for self-learned classic aversive conditioning. This aversive information transfer is species dependent. Solitary flies cannot learn this information through social learning, suggesting that this ability is not an innate behavior. Neurons used to process and execute avoidance behavior to escape from electrically shocked flies are all in the same brain region, indicating that the fly brain has a common center for integrating external stimuli with internal states to generate flight behavior.

IMD signaling in the gut and the brain modulates Amyloid-beta-induced deficits in Drosophila.

AIMS: Evidence indicates accumulating Aß peptides in brain activates immune responses in neuronal and peripheral system, which may collaboratively influence pathogenesis of Alzheimer's disease (AD). We aim to investigate whether regulating intestinal innate immune signaling ameliorates Aß-induced impairments in Drosophila melanogaster. MAIN METHODS: Quantitative polymerase chain reaction (qPCR) was used to observe expression changes of innate immune responses related genes in brain and in gut under the circumstance of Aß overexpressing in nerve system. Aversive olfactory conditioning and survival assay were used to investigate effects of modulating Attacin-A (AttA) and Dpitercin-A (DptA). Fluorometric assays of respiratory burst activity was introduced to explore whether reducing oxidative stress enables overexpressing intestinal AttA and DptA to reverse Aß-induced deficits. KEY FINDINGS: In vivo genetic analysis revealed that accumulating Aß42 in neurons modulates innate immune signaling of the IMD pathway both in the brain and in the gut. Increased expression levels of the intestinal AttA and DptA improved learning performance and extended the lifespan of Aß42 flies. The administration of apramycin led to alleviations of Aß-induced behavioral changes, indicating that gram-negative bacteria are associated with the development of Aß-induced pathologies. Further analysis showed that the neural expression of Aß42 increased oxidative stress in the gut, which disrupted intestinal integrity and decreased learning performance. In addition, increased levels of AMPs targeting gram-negative bacteria and antioxidants reduced oxidative stress in the gut and reversed Aß-induced behavioral damage. SIGNIFICANCE: These findings suggest that innate immune responses in the gut play a pivotal role in modulating Aß-induced pathologies.

Temporal focusing multiphoton microscopy with cross-modality multi-stage 3D U-Net for fast and clear bioimaging.

Temporal focusing multiphoton excitation microscopy (TFMPEM) enables fast widefield biotissue imaging with optical sectioning. However, under widefield illumination, the imaging performance is severely degraded by scattering effects, which induce signal crosstalk and a low signal-to-noise ratio in the detection process, particularly when imaging deep layers. Accordingly, the present study proposes a cross-modality learning-based neural network method for performing image registration and restoration. In the proposed method, the point-scanning multiphoton excitation microscopy images are registered to the TFMPEM images by an unsupervised U-Net model based on a global linear affine transformation process and local VoxelMorph registration network. A multi-stage 3D U-Net model with a cross-stage feature fusion mechanism and self-supervised attention module is then used to infer in-vitro fixed TFMPEM volumetric images. The experimental results obtained for in-vitro drosophila mushroom body (MB) images show that the proposed method improves the structure similarity index measures (SSIMs) of the TFMPEM images acquired with a 10-ms exposure time from 0.38 to 0.93 and 0.80 for shallow- and deep-layer images, respectively. A 3D U-Net model, pretrained on in-vitro images, is further trained using a small in-vivo MB image dataset. The transfer learning network improves the SSIMs of in-vivo drosophila MB images captured with a 1-ms exposure time to 0.97 and 0.94 for shallow and deep layers, respectively.

Early Aß42 Exposure Causes Learning Impairment in Later Life.

Amyloid cascade hypothesis proposes that amyloid ß (Aß) accumulation is the initiator and major contributor to the development of Alzheimer's disease (AD). However, this hypothesis has recently been challenged by clinical studies showing that reduction of Aß accumulation in the brain does not accompany with cognitive improvement, suggesting that therapeutically targeting Aß in the brain may not be sufficient for restoring cognitive function. Since the molecular mechanism underlying the progressive development of cognitive impairment after Aß clearance is largely unknown, the reason of why there is no behavioral improvement after Aß clearance remains elusive. In the current study, we demonstrated that transient Aß expression caused learning deficit in later life, despite the accumulated Aß was soon being removed after the expression. Early Aß exposure decreased the cellular expression of XBP1 and both the antioxidants, catalase, and dPrx5, which made cells more vulnerable to oxidative stress in later life. Early induction of XBP1, catalase, and dPrx5 prevented the overproduction of ROS, improved the learning performance, and preserved the viability of cells in the later life with the early Aß induction. Treating the early Aß exposed flies with antioxidants such as vitamin E, melatonin and lipoic acid, after the removal of Aß also preserved the learning ability in later life. Taken together, we demonstrated that early and transient Aß exposure can have a profound impact on animal behavior in later life and also revealed the cellular and molecular mechanism underlying the development of learning impairment by the early and transient Aß exposure.

Disrupted cellular calcium homeostasis is responsible for Aß-induced learning and memory damage and lifespan shortening in a model of Aß transgenic fly.

Accumulated Aß is one of the hallmarks of Alzheimer's disease. Although accumulated results from in vivo and in vitro studies have shown that accumulated Aß causes learning and memory deficit, cell death, and lifespan reduction, the underlying mechanism remains elusive. In neurons, calcium dynamics is regulated by voltage-gated calcium channel (VGCC) and endoplasmic reticulum and is important for neuron survival and formation of learning and memory. The current study employs in vivo genetics to reveal the role of calcium regulation systems in Aß-induced behavioral damage. Our data shows that although increased VGCC improves learning and memory in Aß42 flies, reduction of VGCC and Inositol trisphosphate receptors extends Aß42 flies' lifespan and improves cell viability. The complex role of calcium regulation systems in Aß-induced damage suggests that the imbalance of calcium dynamic is one of the main factors to trigger learning and memory deficit and cell death in the disease.

The double-edged sword effect of HDAC6 in Aß toxicities.

Alzheimer's disease (AD) is marked by cognitive impairment, massive cell death, and reduced life expectancy. Pathologically, accumulated beta-amyloid (Aß) aggregates and hyperphosphorylated tau protein is the hallmark of the disease. Although changes in cellular function and protein accumulates have been demonstrated in many different AD animal models, the molecular mechanism involved in different cellular functions and the correlation and causative relation between different protein accumulations remain elusive. Our in vivo genetic studies revealed that the molecular mechanisms involved in memory loss and lifespan shortening are different and that tau plays an essential role in mediating Aß-induced early death. We found that when the first deacetylase (DAC) domain of histone deacetylase 6 (HDAC6) activity was increased, it regulated cortactin deacetylation to reverse Aß-induced learning and memory deficit, but with no effect on the lifespan of the Aß flies. On the other hand, an increased amount of the second DAC domain of HDAC6 promoted tau phosphorylation to facilitate Aß-induced lifespan shortening without affecting learning performance in the Aß flies. Our data also confirmed decreased acetylation in two major HDAC6 downstream proteins, suggesting increased HDAC6 activity in Aß flies. Our data established the double-edged sword effect of HDAC6 activity in Aß-induced pathologies. Not only did we segregate memory loss and lifespan shortening in Aß flies, but we also provided evidence to link the Aß with tau signaling.

Rac1 and Akt Exhibit Distinct Roles in Mediating Aß-Induced Memory Damage and Learning Impairment.

Accumulated beta-amyloid (Aß) in the brain is the hallmark of Alzheimer's disease (AD). Despite Aß accumulation is known to trigger cellular dysfunctions and learning and memory damage, the detailed molecular mechanism remains elusive. Recent studies have shown that the onset of memory impairment and learning damage in the AD animal is different, suggesting that the underlying mechanism of the development of memory impairment and learning damage may not be the same. In the current study, with the use of Aß42 transgenic flies as models, we found that Aß induces memory damage and learning impairment via differential molecular signaling pathways. In early stage, Aß activates both Ras and PI3K to regulate Rac1 activity, which affects mostly on memory performance. In later stage, PI3K-Akt is strongly activated by Aß, which leads to learning damage. Moreover, reduced Akt, but not Rac1, activity promotes cell viability in the Aß42 transgenic flies, indicating that Akt and Rac1 exhibit differential roles in Aß regulating toxicity. Taken together, different molecular and cellular mechanisms are involved in Aß-induced learning damage and memory decline; thus, caution should be taken during the development of therapeutic intervention in the future.

Mushroom body subsets encode CREB2-dependent water-reward long-term memory in Drosophila.

Long-term memory (LTM) formation depends on the conversed cAMP response element-binding protein (CREB)-dependent gene transcription followed by de novo protein synthesis. Thirsty fruit flies can be trained to associate an odor with water reward to form water-reward LTM (wLTM), which can last for over 24 hours without a significant decline. The role of de novo protein synthesis and CREB-regulated gene expression changes in neural circuits that contribute to wLTM remains unclear. Here, we show that acute inhibition of protein synthesis in the mushroom body (MB) αß or γ neurons during memory formation using a cold-sensitive ribosome-inactivating toxin disrupts wLTM. Furthermore, adult stage-specific expression of dCREB2b in αß or γ neurons also disrupts wLTM. The MB αß and γ neurons can be further classified into five different neuronal subsets including αß core, αß surface, αß posterior, γ main, and γ dorsal. We observed that the neurotransmission from αß surface and γ dorsal neuron subsets is required for wLTM retrieval, whereas the αß core, αß posterior, and γ main are dispensable. Adult stage-specific expression of dCREB2b in αß surface and γ dorsal neurons inhibits wLTM formation. In vivo calcium imaging revealed that αß surface and γ dorsal neurons form wLTM traces with different dynamic properties, and these memory traces are abolished by dCREB2b expression. Our results suggest that a small population of neurons within the MB circuits support long-term storage of water-reward memory in Drosophila.

Electrical synapses between mushroom body neurons are critical for consolidated memory retrieval in Drosophila.

Electrical synapses between neurons, also known as gap junctions, are direct cell membrane channels between adjacent neurons. Gap junctions play a role in the synchronization of neuronal network activity; however, their involvement in cognition has not been well characterized. Three-hour olfactory associative memory in Drosophila has two components: consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM). Here, we show that knockdown of the gap junction gene innexin5 (inx5) in mushroom body (MB) neurons disrupted ARM, while leaving ASM intact. Whole-mount brain immunohistochemistry indicated that INX5 protein was preferentially expressed in the somas, calyxes, and lobes regions of the MB neurons. Adult-stage-specific knockdown of inx5 in αß neurons disrupted ARM, suggesting a specific requirement of INX5 in αß neurons for ARM formation. Hyperpolarization of αß neurons during memory retrieval by expressing an engineered halorhodopsin (eNpHR) also disrupted ARM. Administration of the gap junction blocker carbenoxolone (CBX) reduced the proportion of odor responsive αß neurons to the training odor 3 hours after training. Finally, the α-branch-specific 3-hour ARM-specific memory trace was also diminished with CBX treatment and in inx5 knockdown flies. Altogether, our results suggest INX5 gap junction channels in αß neurons for ARM retrieval and also provide a more detailed neuronal mechanism for consolidated memory in Drosophila.

Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

Mushroom body glycolysis is required for olfactory memory in Drosophila.

Glucose catabolism, also known as glycolysis, is important for energy generation and involves a sequence of enzymatic reactions that convert a glucose molecule into two pyruvate molecules. The glycolysis process generates adenosine triphosphate as a byproduct. In this study, we investigated whether glycolysis plays a role in maintaining neuronal functions in the Drosophila mushroom bodies (MBs), which are generally accepted to be an olfactory learning and memory center. Our data showed that individual knockdown of glycolytic enzymes in the MBs, including hexokinase (HexA), phosphofructokinase (Pfk), or pyruvate kinase (PyK), disrupts olfactory memory. Whole-mount brain immunostaining indicated that pyruvate kinase is strongly expressed in the MB αß, α'ß', and γ neuron subsets. We conclude that HexA, Pfk, and PyK are required in each MB neuron subset for olfactory memory formation. Our data therefore indicates that glucose catabolism in the MBs is important for olfactory memory formation in Drosophila.

XBP1 and PERK Have Distinct Roles in Aß-Induced Pathology.

Endoplasmic reticulum (ER) stress triggers multiple cellular signals to restore cellular function or induce proapoptosis that is altered in the brains of patients with Alzheimer's disease (AD). However, the role of ER stress in ß-amyloid (Aß)-induced AD pathology remains elusive, and data obtained from different animal models and under different experimental conditions are sometimes controversial. The current study conducted in vivo genetic experiments to systematically examine the distinct role of each ER stress effector during disease progression. Our results indicated that inositol-requiring enzyme 1 was activated before protein kinase RNA-like endoplasmic reticulum kinase (PERK) activation in Aß42 transgenic flies. Proteasome activity played a key role in this sequential activation. Furthermore, our study separated learning deficits from early degeneration in Aß-induced impairment by demonstrating that X-box binding protein 1 overexpression at an early stage reversed Aß-induced early death without affecting learning performance in the Aß42 transgenic flies. PERK activation was determined to only enhance Aß-induced learning deficits. Moreover, proteasome overactivation was determined to delay PERK activation and improve learning deficits. Altogether, the findings of this study demonstrate the complex roles of ER stress during Aß pathogenesis and the possibility of using different ER stress effectors as reporters to indicate the status of disease progression.

Dysfunction of different cellular degradation pathways contributes to specific ß-amyloid42-induced pathologies.

The endosomal-lysosomal system (ELS), autophagy, and ubiquitin-proteasome system (UPS) are cellular degradation pathways that each play a critical role in the removal of misfolded proteins and the prevention of the accumulation of abnormal proteins. Recent studies on Alzheimer's disease (AD) pathogenesis have suggested that accumulation of aggregated ß-amyloid (Aß) peptides in the AD brain results from a dysfunction in these cellular clearance systems. However, the specific roles of these pathways in the removal of Aß peptides and the pathogenesis underlying AD are unclear. Our in vitro and in vivo genetic approaches revealed that ELS mainly removed monomeric ß-amyloid42 (Aß42), while autophagy and UPS clear oligomeric Aß42. Although overproduction of phosphatidylinositol 4-phosphate-5 increased Aß42 clearance, it reduced the life span of Aß42 transgenic flies. Our behavioral studies further demonstrated impaired autophagy and UPS-enhanced Aß42-induced learning and memory deficits, but there was no effect on Aß42-induced reduction in life span. Results from genetic fluorescence imaging showed that these pathways were damaged in the following order: UPS, autophagy, and finally ELS. The results of our study demonstrate that different degradation pathways play distinct roles in the removal of Aß42 aggregates and in disease progression. These findings also suggest that pharmacologic treatments that are designed to stimulate cellular degradation pathways in patients with AD should be used with caution.-Ji, X.-R., Cheng, K.-C., Chen, Y.-R., Lin, T.-Y., Cheung, C. H. A., Wu, C.-L., Chiang, H.-C. Dysfunction of different cellular degradation pathways contributes to specific ß-amyloid42-induced pathologies.

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.

Hemi-fused structure mediates and controls fusion and fission in live cells.

Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic ß-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles.

Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

Exocytosis and endocytosis: modes, functions, and coupling mechanisms.

Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis.

Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.

The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca(2+) influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca(2+) influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.